1. Field of the Invention
The present invention is related to a method for purifying bioactive substances.
2. Description of the Related Art
Obtainment of highly pure proteins, which are targets of research, is extremely important in the academic fields such as molecular biology and biochemistry, as well as in the industrial fields such as biochemistry and pharmaceuticals. Recently, utilization of large scale expression systems that employ recombinant genes and host cells to obtain desired proteins as recombined proteins has become possible. A technique that utilizes so called added tags when purifying proteins using the large scale expression systems, is known.
A technique that purifies proteins by employing portions called tags, which are introduced to the N or C ends of proteins that are artificially synthesized by altering genes, is known. A purification method that employs His-tag is a representative example of this technique. His-tag proteins are proteins that have short peptides with six to ten histidine groups attached to the ends thereof. Because the molecular weight of the tag portion is small, there is little effect on the activity or the structure of target proteins. Therefore, His-tag proteins are widely utilized in purification and detection of recombined proteins.
Purification of His-tag proteins which have been expressed by gene recombination is performed by the following steps. A sample that contains the His-tag proteins is caused to flow through a column, thereby causing the His-tag proteins to bond with media. Then, contaminants are washed away by a cleansing liquid. Thereafter, the His-tag proteins are recovered, by causing an eluting solvent to flow over the media. Accordingly, it is necessary for the His-tag proteins to have sufficient bonding force such that they are not washed away by the cleansing liquid. On the other hand, it is necessary for the bonding force of the His-tag proteins to be weakened by the eluting solvent. Conventionally, cleansing is performed using a buffer having the same pH as that when the His-tag proteins are bonded to the media, and recovery of the His-tag proteins is performed using a buffer of a different pH. Alternatively, an eluting solvent containing imidazole is used to elute the His-tag proteins (refer to International Patent Publication No. WO98/06739, for example).
However, it is difficult to perform quantitative purification at high purity levels using this purification technique. A factor in the difficulty is due to the fact that when large amounts of cleansing liquid are utilized to completely purify the His-tag proteins that include contaminants, the His-tag proteins are washed away along with the contaminants.
“Stable and Functional Immobilization of Histidine-Tagged proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush”, Anal. Chem., Vol. 77, pp. 1096-1105, 2005 discloses a technique for immobilizing His-tag proteins using NTA-Ni(II) complexes. In this technique, water molecules, which are coordinately bonded to two coordinate sites in each complex, are substituted by nitrogen atoms of two imidazole groups of oligohistidine groups of the His-tag proteins. Thereby, His-tag proteins are caused to specifically bind to the surface of media.
However, His-tag proteins cannot be stably immobilized even if the technique disclosed in “Stable and Functional Immobilization of Histidine-Tagged proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush”, Anal. Chem., Vol. 77, pp. 1096-1105, 2005 is applied, if large amounts of cleansing liquid are used. In addition, there is a problem that this technique is not practical from the viewpoint of recovery rates of protein, because the amount of proteins that can be held is small.